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Engineering the redox potential over a wide range within a new class of FeS proteins.

Zuris JA, Halim DA, Conlan AR, Abresch EC, Nechushtai R, Paddock ML, Jennings PA - J. Am. Chem. Soc. (2010)

Bottom Line: Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins.We tested the robustness of mitoNEET to mutation and the range over which the redox potential (E(M)) could be tuned.We found that the protein could tolerate an array of mutations that modified the E(M) of the [2Fe-2S] center over a range of ∼700 mV, which is the largest E(M) range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093, USA.

ABSTRACT
MitoNEET is a newly discovered mitochondrial protein and a target of the TZD class of antidiabetes drugs. MitoNEET is homodimeric with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry. Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins. We tested the robustness of mitoNEET to mutation and the range over which the redox potential (E(M)) could be tuned. We found that the protein could tolerate an array of mutations that modified the E(M) of the [2Fe-2S] center over a range of ∼700 mV, which is the largest E(M) range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV). These properties make mitoNEET potentially useful for both physiological studies and industrial applications as a stable, water-soluble, redox agent.

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(A) Structure near the [2Fe-2S] center (Fe atoms as red spheres, sulfur as yellow) of mitoNEET showing both the ligating and nearby (<5 Å) titratable residues. (B) Representative pH dependences of the EM for select mutants. WT, D84G, and D84N exhibit similar dependences (−51 mV/pH) at pH ≥ 7, implying that, in these cases, reduction remains proton-coupled. H87C shows a more shallow slope (−15 mV/pH) for the pH-dependence,(10) indicating that His87 is principally responsible for the observed proton coupling in WT. Replacement of Lys55 with Glu (K55E) shifts the pKox of His87 from 6.7 ± 0.2 to 9.2 ± 0.2.
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fig3: (A) Structure near the [2Fe-2S] center (Fe atoms as red spheres, sulfur as yellow) of mitoNEET showing both the ligating and nearby (<5 Å) titratable residues. (B) Representative pH dependences of the EM for select mutants. WT, D84G, and D84N exhibit similar dependences (−51 mV/pH) at pH ≥ 7, implying that, in these cases, reduction remains proton-coupled. H87C shows a more shallow slope (−15 mV/pH) for the pH-dependence,(10) indicating that His87 is principally responsible for the observed proton coupling in WT. Replacement of Lys55 with Glu (K55E) shifts the pKox of His87 from 6.7 ± 0.2 to 9.2 ± 0.2.

Mentions: EM values of WT and mutant mitoNEET have been engineered over a range of ∼700 mV and can be tuned to obtain nearly any value within the EM range shown. Measurements adjusted to SHE values, with errors (±10 mV) indicated by cross bars. *EM extremes (see Figure 3B). The EM values of several cellular environments are shown on the left bar. Abbreviations: Resp. (Respiratory tract fluid, +200 mV), β-Cell (Cytoplasm of beta cells, producers of insulin in the pancreas, +55 mV), Cyt (Cytoplasm, −205 mV), ER (Endoplasmic Reticulum, −217 mV), Mito (Mitochondria, −260 mV).(4)


Engineering the redox potential over a wide range within a new class of FeS proteins.

Zuris JA, Halim DA, Conlan AR, Abresch EC, Nechushtai R, Paddock ML, Jennings PA - J. Am. Chem. Soc. (2010)

(A) Structure near the [2Fe-2S] center (Fe atoms as red spheres, sulfur as yellow) of mitoNEET showing both the ligating and nearby (<5 Å) titratable residues. (B) Representative pH dependences of the EM for select mutants. WT, D84G, and D84N exhibit similar dependences (−51 mV/pH) at pH ≥ 7, implying that, in these cases, reduction remains proton-coupled. H87C shows a more shallow slope (−15 mV/pH) for the pH-dependence,(10) indicating that His87 is principally responsible for the observed proton coupling in WT. Replacement of Lys55 with Glu (K55E) shifts the pKox of His87 from 6.7 ± 0.2 to 9.2 ± 0.2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944382&req=5

fig3: (A) Structure near the [2Fe-2S] center (Fe atoms as red spheres, sulfur as yellow) of mitoNEET showing both the ligating and nearby (<5 Å) titratable residues. (B) Representative pH dependences of the EM for select mutants. WT, D84G, and D84N exhibit similar dependences (−51 mV/pH) at pH ≥ 7, implying that, in these cases, reduction remains proton-coupled. H87C shows a more shallow slope (−15 mV/pH) for the pH-dependence,(10) indicating that His87 is principally responsible for the observed proton coupling in WT. Replacement of Lys55 with Glu (K55E) shifts the pKox of His87 from 6.7 ± 0.2 to 9.2 ± 0.2.
Mentions: EM values of WT and mutant mitoNEET have been engineered over a range of ∼700 mV and can be tuned to obtain nearly any value within the EM range shown. Measurements adjusted to SHE values, with errors (±10 mV) indicated by cross bars. *EM extremes (see Figure 3B). The EM values of several cellular environments are shown on the left bar. Abbreviations: Resp. (Respiratory tract fluid, +200 mV), β-Cell (Cytoplasm of beta cells, producers of insulin in the pancreas, +55 mV), Cyt (Cytoplasm, −205 mV), ER (Endoplasmic Reticulum, −217 mV), Mito (Mitochondria, −260 mV).(4)

Bottom Line: Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins.We tested the robustness of mitoNEET to mutation and the range over which the redox potential (E(M)) could be tuned.We found that the protein could tolerate an array of mutations that modified the E(M) of the [2Fe-2S] center over a range of ∼700 mV, which is the largest E(M) range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093, USA.

ABSTRACT
MitoNEET is a newly discovered mitochondrial protein and a target of the TZD class of antidiabetes drugs. MitoNEET is homodimeric with each protomer binding a [2Fe-2S] center through a rare 3-Cys and 1-His coordination geometry. Both the fold and the coordination of the [2Fe-2S] centers suggest that it could have novel properties compared to other known [2Fe-2S] proteins. We tested the robustness of mitoNEET to mutation and the range over which the redox potential (E(M)) could be tuned. We found that the protein could tolerate an array of mutations that modified the E(M) of the [2Fe-2S] center over a range of ∼700 mV, which is the largest E(M) range engineered in an FeS protein and, importantly, spans the cellular redox range (+200 to -300 mV). These properties make mitoNEET potentially useful for both physiological studies and industrial applications as a stable, water-soluble, redox agent.

Show MeSH
Related in: MedlinePlus